Somatic Embryogenesis Of Hippophae Rhamnoides L. And Related To Study On Genetic Transformation Of Arabidopsis Thaliana (Linn.) Heynh. With BADH

As a kind of multi-function economic tree, seabuckthorn (Hippophae rhamnoides L.) is distributed widely in Asia-Europe, which has some important values in the field of ecology, medicine, food and so on. But now, there were some limiting factors in seabuckthorn propagation, that results in a low output about seabuckthorn choiceness seedlings and could not meet the great demands of markets. In the recent years, there is a very serious problem about seabuckthorn, so lots of experts gave a kind of unanimous analysis about the phenomenon, that was low anti- adversity ability effecting on inferior growth of seabuckthorn, and genetic transformation method of anti-adversity gene maybe the best choice to settle problem. The regeneration system by indirection somatic embryogenesis, not only is an effective technique means to get a number of choiceness seedlings, but also give a good base to have related to genetic transformation research in seabuckthorn. As an important economic fruit tree, seabuckthorn are applied widely in the food, medicine and feedstuff field. If BADH gene as aim gene and marker gene at the same time is transferred into plant, not only transformation tree can get high anti-adversity ability, but also the latency harm problem can be avoided from resistance marker gene. So, it is very significative to develop BADE gene as marker gene in genetic transformation research.During two years, on the base of reported research, the regeneration system of indirected somatic embryogenesis in seabuckthorn was achieved and there were some discussions and analyses about related factors of somatic embryogenesis. In genetic transformation research of Arabidopsis thaliana,some phenotypic difference were discussed about using BADH as marker gene to screen positive plants and those work also can offer strong supports for next researches. The results were as follows:1.During experiment of callus and embryogenic callus induction using shoot tips from tissue culture plant of seabuckthorn as explants, callus incidence rate was the highest on 1/4MS medium, which had key function to induce callus. Either using NAA or 2,4-D alone, embryogenic callus could not be induced, indicating that the combination of auxin and cytokinin is necessary in the process of inducing embryogenic callus. On the same time, a conclusion was summarized that the combination of NAA and KT was effective to embryogenic callus induction in seabuckthorn.2.On the base of experiment of callus and embryogenic callus induction in seabuckthorn, some factors were discussed related to embryogenic callus initiation. Finally, the achievements were as follows: the induction frequency of callus reached 90.10%, the induction frequency of embryogenic callus was 77.67% and the average number of somatic embryogenesis could up to 15.48. So the suitable medium to inducing embryogenic callus and somatic embryos was 1/4MS +KT1.0mg/L +NAA0.3mg/L +granulated sugar 30g/L +agar6.5g/L +CH0.5g/L.3.By the research about different inoculation manners of seabuckthorn cotyledon and hypocotyl on inducing medium, the result showed that different inoculation manners could affect inducing effect of callus and embryogenic callus, and the inducing rate from high to low in turn about cotyledon was cotyledon incision downside >abaxial side downside > adaxial side downside, and the under side inoculation of inclined hypocotyl was better than horizontal placement.4.The research results about shoot tip, cotyledon, hypocotyl in seabuckthorn were inoculated respectively on inducing medium, indicated that the effect of shoot tip was better than cotyledon and hypocotyl, which had 90.10% callus inducing rate and 77.67% embryogenic callus inducing rate. Otherwise, the callus inducing rate and embryogenic callus inducing rate of cotyledon were only 66.73% and 14.80%, respectively, and the callus inducing rate and embryogenic callus inducing rate of hypocotyls were 52.80% and 34.87%, respectively. The inducing rate of both cotyledon and hypocotyl were all very lower than shoot tip's. As a result, shoot tip was the best explant during the inducing embryogenic callus experiment.5.By experiment of the plant regeneration from somatic embryo in seabuckthorn under the effect of KT and basal medium, WPM had the best effect on promoting few plants regeneration in seabuckthorn among MS, 1/2MS 1/4MS and WPM mediums. Meanwhile, reducing granulated sugar and design different concentrations KT were used to have the experiment of plant regeneration. Finally, the best regeneration medium was:WPM +KT 0.05mg/L +granulated sugar 20g/L +agar6.5g/L +CH0.5g/L. And regeneration frequency was 60.30% on the medium.6.Using Trizol method to extract RNA from spinach, using DNAMAN sofeware to design primer BH4.24 1:5'-CTCGCCTTACCCTCTCAACTC -3' and BH4.24.2:5'-CTGT AAACTCGACCTTCTCTGCG -3'and using RT-PCR method to clone BADH gene, the sequence was cloned successfully by DNA Sequencing analysis.7.After restriction sites Bglâ…¡(agatct) and Pmlâ… (cacgtg) were added successfully to upstream and downstream of the cloned BADH sequence from spinach, the expression vector pCAMBIA1305.2-BADH was constructed and transformed in Arabidopsis thaliana subsequently.8.Because of both BADH gene and hygromycin resistance gene existing on the expression vector pCAMBIA1305.2-BADH,transformed plant could be selected using BA' and hygromycin, which had a function to validate each other. The results showed that transformed plant was not selected on 1/2MS supplemented with 20mM BA'.On the other hand, four transformed plant lines (HBT₀(1),HBT₀(2),HBT₀(3),HBT₀(4)) and four control plant lines (HKT₀(1),HKT₀(2), HKT₀(3),HKT₀(4)) were got on 1/2MS supplemented with 25mg/L hygromycin, respectively.9.By PCR, transformed plant lines with BADH (HBT₀(1),HBT₀(2)) was detection, the test result showed BADH gene had been transformed into Arabidopsis thaliana DNA genome. After T₁ generation seeds were selected on selective medium supplemented with 25mg/L hygromycin, there was a 3:1 Mendelian's segregation law between transgene and non-transgene plants.10.T₁ generation positive plants and mature seeds from transgenic plant lines with BADH (HBT₀(1),HBT₀(2)) were cultured on different concentrations NaCl mediums in the condition of light and night, root and hypocotyl were affected at different extent and exhibited some anti-NaCl ability, especially at the phenotypic analyse of positive plants, transformed plant lines with BADH could survived and grew strongly than control plant lines on 100mM NaCl medium, indicating transformed plant could suffer the stress treatment of 100mM NaCl.11.T₁ generation positive plants and mature seeds from transgenic plant lines with BADH (HBT₀(1),HBT₀(2)) were cultured on 20mM BA' medium on the condition of light and night, the phenotypic characters of transformed plant with BADH, such as root and hypocotyl were longer than control plants, which indicated some resistance function in transformed plant lines, especially at the phenotypic analyse of positive plants, the difference of root was the best obvious between transgenic plants with BADH and control plants, which was consistent with the conclusion of sensitivity test and further verified the feasibility of using the root phenotypic characters to screen positive plants with BADH in Arabidopsis thaliana. On the side, by right of the selective standard in root length,15 days was the suitable time to judge transgenic plant lines, which offered some phenotypic selectable characters to select transformed Arabidopsis thaliana by BA' selective agent.